Mass spectrometry (MS) is an analytical technique to filter, detect, identify and/or measure compounds by the mass-to-charge ratios of ions formed from the compounds. The quantity of mass-to-charge ratio is commonly denoted by the symbol “m/z” in which “m” is ionic mass in units of Daltons and “z” is ionic charge in units of elementary charge, e. Thus, mass-to-charge ratios are appropriately measured in units of “Da/e”. Mass spectrometry techniques generally include (1) ionization of compounds and optional fragmentation of the resulting ions so as to form fragment ions; and (2) detection and analysis of the mass-to-charge ratios of the ions and/or fragment ions and calculation of corresponding ionic masses. The compound may be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer and an ion detector.
The hybrid technique of liquid chromatography-mass spectrometry (LC/MS) is an extremely useful technique for detection, identification and (or) quantification of components of mixtures or of analytes within mixtures. This technique generally provides data in the form of a mass chromatogram, in which detected ion intensity (a measure of the number of detected ions) as measured by a mass spectrometer is given as a function of time. In the LC/MS technique, various separated chemical constituents elute from a chromatographic column as a function of time. As these constituents come off the column, they are submitted for mass analysis by a mass spectrometer. The mass spectrometer accordingly generates, in real time, detected relative ion abundance data for ions produced from each eluting analyte, in turn. Thus, such data is inherently three-dimensional, comprising the two independent variables of time and mass (more specifically, a mass-related variable, such as mass-to-charge ratio) and a measured dependent variable relating to ion abundance. The term “liquid chromatography” includes, without limitation, reverse phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), normal-phase high performance liquid chromatography (NP-HPLC), supercritical fluid chromatography (SFC) and ion chromatography.
Conventionally, one can often enhance the resolution of the MS technique by employing “tandem mass spectrometry” or “MS/MS”, via use, for example, of a triple quadrupole mass spectrometer. In this technique, a first (or parent or precursor) ion species generated from a molecular species of interest can be filtered or isolated in an MS instrument. The precursor ions of the various precursor ion species can be subsequently fragmented to yield one or more second (or product or fragment) ions comprising various product/fragment ion species that are then analyzed in a second MS stage. By careful selection of precursor ion species, only ions produced by certain analytes are passed to the fragmentation chamber or other reaction cell, such as a collision cell where collision of ions with atoms of an inert gas produces the product ions.
Typically, mass spectral experiments that survey for the presence of multiple analytes using the MS/MS technique rely on automatic data-dependent decision logic. Data-dependent acquisition involves using data derived from an experimentally-acquired mass spectrum in an “on-the-fly” manner to direct the subsequent operation of a mass spectrometer; for example, a mass spectrometer may be switched between MS and MS/MS scan modes upon detection of an ion species of potential interest. Utilization of data-dependent acquisition methods in a mass spectrometer provides the ability to make automated, real-time decisions in order to maximize the useful information content of the acquired data.
More generally, data-dependent acquisition methods may be characterized as having one or more input criteria, and one or more output actions. The input criteria employed for conventional data-dependent methods are generally based on parameters such as intensity, intensity pattern, mass window, mass difference (neutral loss), mass-to-charge (m/z) inclusion and exclusion lists, and product ion mass. The input criteria are employed to select one or more ion species that satisfy the criteria. The selected ion species are then subjected to an output action (examples of which include performing MS/MS or MSn analysis and/or high-resolution scanning).
Unfortunately, the identification of compounds by either the MS or MS/MS technique may, in practice, be complicated by the fact that any single chemical compound can give rise to many mass spectral ionic species in a typical LC-MS analysis. The multiple mass spectral species result from the formation of more than one adducted and polymer ion species from each compound. Additionally, each such ion species will generally give rise to multiple mass spectral peaks because of the presence of multiple isotopes of various constituent elements as well as the existence of polymerization and multiple charge states. Adduction of the molecule being analyzed can involve combinations of various additions or losses of charged (e.g. H+, Na+, NH4+, K+, . . . ) and neutral (H2O, MeOH, ACN, . . . ) atom groups. Typically, determination of adducts and polymers and, to a lesser extent, charge states of small molecules requires the assignment of the monoisotopic (M+H)+ or (M−H)− species for which then differences to other species are calculated and determined. Failure to properly recognize or account for multiple related mass spectral peaks resulting from a single compound may cause undesirable false positive identifications of certain analytes or lead to incorrect selection of precursor ions during a data-dependent decision step of an MS/MS analysis.